A subpopulation of rat glial cells, derived from the astroglial population of newborn cerebral cortex cell cultures, spontaneously transformed in culture. Unlike the pretransformed cells, the transformed cells formed pile-up colonies, exhibited anchorage-independent growth, and were tumorigenic in young rats. Both the pretransformed and transformed cells exhibited differentiated properties characteristic of glial cells. For example, the pretransformed cells possessed hydrocortisone-inducible glutamine synthetase (GS), a property restricted to astrocytes in the central nervous system. As was anticipated, these cells did not exhibit either of two oligodendroglial characteristics, hydrocortisone-inducible glycerol phosphate dehydrogenase (GPDH) or the induction of lactate dehydrogenase (LDH) by N6,O6-dibutyryl cyclic adenosine 3':5'monophosphate (Bt2 cAMP). Unexpectedly, the transformed cells expressed the induction of glycerol phosphate dehydrogenase and lactate dehydrogenase but lost the glutamine synthetase induction. Both the pretransformed and transformed cells were examined ultrastructurally. Neither cell type exhibited glial filaments (9-10 nm), a structure typical of astrocytes. Rather, the pretransformed cells were characterized by distinct longitudinal filaments near the cell surface and the absence of microtubules. On the other hand, the only cytoskeletal element visible in transformed cells were microtubules. Our work demonstrates that, like other rodent cell types, rat glial cells can spontaneously transform in culture. It also shows that the expression of differentiated properties are sensitive to the transformation process.